GSM5603402: 4_20_3, eccD1, M63 + NH4Cl; Mycolicibacterium smegmatis MC2 155; RNA-Seq
Cultures were harvested by centrifugation,  pellets were re-suspended in RNA Protect buffer from the Monarch Total RNA Miniprep Kit. Re-suspended pellets were flash frozen in liquid nitrogen and stored at -80°C. Extraction was done by  re-suspending frozen pellets in kit lysis buffer and 0.1 mm zirconia beads were used to lyse samples with a vortex adapter bead beater, the kit protocol was followed as per the manufacturer's protocol. RNA was DnaseI digested after extraction and rRNA was depleted using the  NEBNext rRNA Depletion Kit (Bacteria). Libraries for RNAseq were prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina and multiplexing was done with custom multiplex oligos. Average fragment sizes were determined using the High Sensitity DNA bioanalyzer chip from Aligent Technologies. Libraries had an average length of 200 - 400 bp. The concentration of the individual libraries was determined by Quibit dsDna High Sensitivity Assay (Invitrogen). 10ng of each livrary was pooled into a final library and ran on a High Sensitivity DNA bioanalyzer chip to determine average fragment size. Final library was submitted to the Chan Zuckerberg Biohub Sequencing platform.